High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses.

Abstract

Abstract. We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAbl08), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a eDNA encoding the full-length human EGF receptor gene, and fully responsive to EGE The binding to saturation of mAbl08 to the surface of these cells at 4°C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGE The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37°C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 106 M-~-s -~) than did low-affinity receptor (2.5 × 105 M-Ls-~), whereas the off-rate constants, measured at 4°C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors.At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAbl08 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca 2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAbl08 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Amphiregulin acts as an autocrine growth factor in two human polarizing colon cancer lines that exhibit domain selective EGF receptor mitogenesis

“…The fact that the EGFR Scatchard plots for cells cultured on plastic were curvilinear suggests that ternary complexes were formed by the receptor, ligand and another protein(s) (Gex-Fabry et al, 1986;Mayo et al, 1989;Wofsy et al, 1992). There are a number of observations that favour the correlation between EGFR affinity and biological effect (Defize et al, 1989;Bellot et al, 1990). Others have found a similar correlation between the affinity state of the EGFR and proliferative effect after growth factor administration (Fowler et al, 1995).…”

Structure-Based View of Epidermal Growth Factor Receptor Regulation

“…Since early studies of EGFR it has been known that there are two affinity "classes" for EGF binding to its cell surface receptor (28,29). These classes are evident in curvilinear Scatchard plots for EGF binding that have been interpreted as indicating a high-affinity (2% -5%) class and a lowaffinity (the remainder) class of sites (3). Several studies attempt to correlate these states with the structure-based model for dimerization of the extracellular region of EGFR with conflicting conclusions (38,47,64).…”

Identification of p130 as an in VivoSubstrate of Receptor Protein-tyrosine Phosphatase α

“…Immunoprecipitation of HA-RPTP␣ was done by incubation with anti-hemagglutinin epitope tag antibody (mAb 12CA5) and protein A-Sepharose (Amersham Pharmacia Biotech). The EGFR was immunoprecipitated using mAb 108.1 (46). cas was precipitated by incubation with glutathione-agarose beads for 3 h at 4°C.…”